This invention relates to medical science particularly the treatment of sepsis with protein C in combination with Bactericidal Permeability Increasing (BPI) Protein.
Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of hemostasis by inactivating Factors Va and VIIIa in the coagulation cascade. Human protein C circulates as a 2-chain zymogen, but functions at the endothelial and platelet surface following conversion to activated protein C (aPC) by limited proteolysis with thrombin in complex with the cell surface membrane protein, thrombomodulin.
In conjunction with other proteins, aPC functions as perhaps the most important down-regulator of blood coagulation resulting in protection against thrombosis. In addition to its anti-coagulation functions, aPC has anti-inflammatory effects through its inhibition of cytokine generation (e.g. TNF and IL-1) and also exerts profibrinolytic properties that facilitate clot lysis. Thus, the protein C enzyme system represents a major physiological mechanism of anti-coagulation, anti-inflammation, and fibrinolysis.
Bactericidal permeability-increasing protein (BPI), is a protein isolated from the granules of mammalian polymorphonuclear neutrophils (PMNs). Human BPI has been isolated from PMNs by acid extraction combined with chromatography (Elsbach, 1979, J. Biol. Chem. 254:11000; Weiss et al. 1987, Blood 69:652), and has been shown to have potent bactericidal activity against a broad spectrum of Gram-negative bacteria. In addition to its bactericidal effect on Gram negative bacteria, BPI is also capable of binding to and neutralizing lipopolysaccharide (LPS) which is also known as endotoxin because of the inflammatory response that it stimulates.
Sepsis, which includes severe sepsis and septic shock, is a systemic inflammatory response to infection or trauma, associated with and mediated by the activation of a number of host defense mechanisms including the cytokine network, leukocytes, and the complement and coagulation/fibrinolysis systems. [Mesters, et al., Blood 88:881-886, 1996]. Disseminated intravascular coagulation [DIC], with widespread deposition of fibrin in the microvasculature of various organs, is an early manifestation of sepsis/septic shock. DIC is an important mediator in the development of the multiple organ failure syndrome and contributes to the poor prognosis of patients with septic shock. [Fourrier, et al., Chest 101:816-823, 1992].
Sepsis may be caused by bacterial (either Gram negative or Gram positive), fungal, viral and other infections as well as by non-infective stimuli such as multiple trauma, severe burns, and organ transplantation.
Although sepsis can follow any bacterial infection, it is often associated with a gram negative infection. Sepsis usually begins with tremor, fever, falling blood pressure, rapid breathing and heart beat, and skin lesions. Within hours or days it can progress to spontaneous clotting in the blood vessels, severe hypotension, multiple organ failure, and death.
Most of-the damage comes not from the invading bacteria but from enotoxin. This effect by endotoxin is manifested by its binding to cells such as monocytes/macrophages or endothelial cells, and triggering them to produce various mediators such as tumor necrosis factor-alpha (TNF-xcex1), and various interleukins (IL-1, IL-6, and IL-8). Production of excessive TNF-xcex1) IL-1, IL-6, and IL-8 can elicit septic shock.
There have been numerous recent attempts to treat sepsis in humans, for the most part using agents that block inflammatory mediators associated with the pathophysiology of this disease. However, clinical studies with a variety of agents that block inflammatory mediators have been unsuccessful [reviewed in Natanson, et al., Ann. Intern. Med 120:771-783, 1994; Gibaldi, Pharmacotherapy 13:302-308, 1993]. Since many of the mediators involved in inflammation are compensatory responses, and therefore have salutary effects, some investigators have suggested that blocking their action may not be appropriate [e.g., Parrillo, N. Engl. J. Med. 328:1471-1477, 1993].
Several encouraging studies using protein C in various animal models of sepsis have been reported. A study in a baboon sepsis model by Taylor, et al., [J. Clin. Invest. 79:918-25, 1987], used plasma-derived human activated protein C. The animals were treated prophylactically (i.e., the aPC was given at the start of the two hour infusion of the LD100 E. coli). Five out of five animals survived 7 days and were considered permanent survivors to the experimental protocol. In control animals receiving an identical infusion of E. coli, five out of five animals died in 24 to 32 hours. In addition, plasma-derived human protein C zymogen has been used as a successful adjunct to aggressive conventional therapy in the management of human patients with purpura fulminans in bacterial sepsis (Gerson, et al., Pediatrics 91:418-422, 1993; Smith, et al., Thromb. Haemost, PS1709, p419, 1997; Rintala, et al., Lancet 347:1767, 1996; Rivard, et al., J. Pediatr. 126:646-652, 1995). 
Recombinant BPI protein has been shown to neutralize lethal and sublethal effects of endotoxin administered to mice, rats, and rabbits (Fisher, et al., Crit. Care Med., 22(4): 553-558, 1994). Because of this ability to neutralize endotoxin and its Gram-negative bactericidal activity, BPI can be utilized for the treatment of human patients suffering from diseases caused by gram-negative bacteria, including bacteremia, endotoxemia, and sepsis.
The present invention is the first to describe the combination of aPC with BPI in the treatment of sepsis. The combination of aPC and BPI results in a synergy that allows the reduction of the dosages of both aPC and BPI and an improvement of clinical outcome of the patient being treated. The reduction of the dosages of the agents in combination therapy in turn results in reduced side effects that may occur with either agent. Therefore, combining aPC, with its anti-coagulant/anti-inflammatory properties, and BPI, with its bactericidal and endotoxin neutralizing activities will provide an effective synergistic therapy for sepsis that will reduce or ameliorate the adverse events and improve the clinical outcome of septic patients.
The present invention provides a method of treating a patient suffering from sepsis which comprises administering to said patient a pharmaceutically effective amount of protein C in combination with bactericidal permeability-increasing (BPI) protein.
The present invention further provides a method of treating sepsis in a patient in need thereof, which comprises administering to said patient a pharmaceutically effective amount of BPI protein and activated protein C such that an activated protein C plasma level of about 2 ng/ml to about 300 ng/ml is achieved.
For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below.
Protein C refers to a vitamin K dependent serine protease with anticoagulant, anti-inflammatory, and profibrinolytic properties which includes, but is not limited to, plasma derived and recombinant produced protein C. Protein C includes and is preferably human protein C although protein C may also include other species or derivatives having protein C proteolytic, amidolytic, esterolytic, and biological (anticoagulant, pro-fibrinolytic, and anti-inflammatory) activities. Examples of protein C derivatives are described by Gerlitz, et al., U.S. Pat. No. 5,453,373, and Foster, et al., U.S. Pat. No. 5,516,650, the entire teachings of which are hereby included by reference.
Zymogenxe2x80x94an enzymatically inactive precursor of a proteolytic enzyme. Protein C zymogen, as used herein, refers to secreted, inactive forms, whether one chain or two chains, of protein C.
Activated protein C or aPC refers to protein C zymogen which has been converted by limited proteolysis to its activated form. aPC includes and is preferably human protein C although aPC may also include other species or derivatives having protein C proteolytic, amidolytic, esterolytic, and biological (anticoagulant or pro-fibrinolytic) activities. Examples of protein C derivatives are noted above in the description of protein C.
r-hPCxe2x80x94recombinant human protein C zymogen.
r-aPCxe2x80x94recombinant activated protein C, preferably produced by activating r-hPC in vitro or by direct secretion of the activated form of protein C from procaryotic cells, eukaryotic cells, and transgenic animals or plants, including, for example, secretion from human kidney 293 cells as a zymogen then purified and activated by techniques well known to the skilled artisan and demonstrated in Yan, U.S. Pat. No. 4,981,952, and Cottingham, WO97/20043, the entire teachings of which are herein incorporated by reference.
Plasma derived activated protein Cxe2x80x94activated protein C produced by activating plasma protein C as described in Eibl, U.S. Pat. No. 5,478,558, the entire teaching of which is herein incorporated by reference.
Continuous infusionxe2x80x94continuing substantially uninterrupted the introduction of a solution into a vein for a specified period of time.
Bolus injectionxe2x80x94the injection of a drug in a defined quantity (called a bolus) over a period of time up to about 120 minutes.
Suitable for administrationxe2x80x94a lyophilized formulation or solution that is appropriate to be given as a therapeutic agent.
Unit dosage formxe2x80x94refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
Pharmaceutically effective amountxe2x80x94represents an amount of protein C of the present invention that is capable of treating sepsis in humans. The particular dose of protein C administered according to this invention will, of course, be determined by the attending physician evaluating the particular circumstances surrounding the case.
BPI proteinxe2x80x94includes naturally and recombinantly produced bactericidal permeability increasing (BPI) protein; natural, synthetic, and recombinant biologically active polypeptide fragments of BPI protein; biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins and dimers; biologically active polypeptide analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs; and BPI-derived peptides. The complete amino acid sequence of human BPI, as well as the nucleotide sequence of DNA encoding BPI have been elucidated by Gray et al., 1989, J. Biol. Chem 264:9505. Recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI are disclosed in U.S. Pat. No. 5,198,541, herein incorporated by reference.
The present invention relates to the treatment of sepsis with protein C in combination with BPI protein. The combination of protein C and BPI results in a synergy that allows the reduction of the dosages of both protein C and BPI and an improvement of clinical outcome of the patient being treated. The reduction of the dosages of the agents in combination therapy in turn results in reduced side effects that may occur with either agent. Therefore, combining protein C, with its anti-coagulant/anti-inflammatory properties, and BPI, with its bactericidal and endotoxin neutralizing activities will provide an effective synergistic therapy for sepsis that will reduce or ameliorate the adverse events and improve the clinical outcome of septic patients.
The protein C administered according to this invention may be generated and/or isolated by any means known in the art or as described in U.S. Pat. No. 4,981,952, and U.S. Pat. No. 5,550,036, herein incorporated by reference. For example, the invention provides a method for producing and secreting full-length, soluble protein C, or biologically active polypeptide variants of protein C from a cell which comprises (a) constructing a vector comprising DNA encoding protein C; (b) transfecting the cell with the vector; and (c) culturing the cell so transfected in culture medium under conditions such that full length soluble protein C or biologically active polypeptide variants of protein C, is secreted. Further, the cell is a eukaryotic cell, e.g. mammalian cell such as Syrian hamster AV12 cell, human embryonic 293 cell, or Baby Hamster Kidney cell.
The protein C used in such combination can be formulated according to known methods to prepare pharmaceutically useful compositions. For example, a desired formulation would be one that is a stable lyophilized product of high purity comprising a bulking agent such as sucrose, a salt such as sodium chloride, a buffer such as sodium citrate and protein C or aPC.
The protein C will be administered parenterally to ensure its delivery into the bloodstream in an effective form by injecting the appropriate dose as continuous infusion for about 1 hour to about 240 hours.
In conjunction with treatment with BPI protein, the amount of protein C administered will be from about 5.0 xcexcg/kg/hr to about 250 xcexcg/kg/hr. Preferably, the protein C administered in combination with BPI protein will be activated protein C. The aPC administered will be from about 1.0 xcexcg/kg/hr to about 50 xcexcg/kg/hr. More preferably the amount of aPC administered will be about 1.0 xcexcg/kg/hr to about 40 xcexcg/kg/hr. While more preferably the amount of aPC administered will be about 1.0 xcexcg/kg/hr to 35 xcexcg/kg/hr. Even more preferably the amount of aPC administered will be about 5.0 xcexcg/kg/hr to 30 xcexcg/kg/hr. Yet even more preferably the amount of aPC administered will be about 15 xcexcg/kg/hr to 30 xcexcg/kg/hr. Still even more preferably the amount of aPC administered will be about 20 xcexcg/kg/hr to 30 xcexcg/kg/hr. The most preferable amount of aPC administered will be about 24 xcexcg/kg/hr. The appropriate dose of aPC administered with BPI protein results in either an improved efficacy or reduction in dose of either agent or both.
The plasma ranges obtained from the amount of aPC administered will be about 2 ng/ml to about 300 ng/ml. The preferred plasma ranges are from about 2 ng/ml to 200 ng/ml. Most preferably, plasma ranges are from about 30 ng/ml to about 150 ng/ml and still more preferably about 100 ng/ml.
Alternatively, the aPC will be administered by injecting one third of the appropriate dose per hour as a bolus injection followed by the remaining two thirds of the hourly dose as continuous infusion for one hour followed by continuous infusion of the appropriate dose for twenty-three hours which results in the appropriate dose administered over 24 hours. In addition, the bolus injection will be administered via an intravenous bag drip pump or syringe pump at about 2 times the normal rate for about 10 to 20 minutes followed by about 1.5 times the normal rate for about 40 to 50 minutes. The normal rate i.e. that rate which has been determined to administer the appropriate dose level of the therapeutic agent per time period, is then continued for up to 240 hours over 24 hours.
BPI protein suitable for use under the present invention includes, but is not limited to, naturally and recombinantly produced BPI protein, for example, a recombinant BPI holoprotein as described in Gray et al. (1989) and U.S. Pat. No. 5,733,872, herein incorporated by reference; natural, synthetic, and recombinant biologically active polypeptide fragments of BPI protein, for example, as described in Ooi et al., J. Exp. Med, 174:649 (1991) and Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992); biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins and dimers; biologically active polypeptide analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs; and BPI-derived peptides, examples of which are described in U.S. Pat. Nos. 5,733,872, 5,627,262, 5,753,620, 5,607,916 and 5,756,464, herein incorporated by reference.
Preferably, the BPI protein of the present invention includes biologically active molecules that have the same or similar amino acid sequence as a natural human BPI holoprotein. Nonlimiting examples of such BPI proteins are the 25 Kd N-terminal fragment of natural human BPI protein, described in Ooi et al., (1991), and the recombinant expression product of DNA encoding N-terminal amino acids from 1 to about 193 or 199 of natural human BPI, described in Gazzano-Santoro et al., (1992).
The BPI protein administered according to this invention may be generated and/or isolated by any means known in the art or as described in U.S. Pat. No. 5,308,834, herein incorporated by reference. For example, the invention provides a method for producing and secreting full-length, soluble BPI holoprotein, biologically active polypeptide fragments, or biologically active polypeptide variants of BPI protein or fragments thereof from a cell which comprises (a) constructing a vector comprising DNA encoding BPI; (b) transfecting the cell with the vector; and (c) culturing the cell so transfected in culture medium under conditions such that full length soluble BPI protein, biologically active polypeptide fragments, or biologically active polypeptide variants of BPI protein or fragments thereof, is secreted. Further, the cell is a eukaryotic cell, e.g. mammalian cell such as Syrian hamster AV12 cell, human embryonic 293 cell, or Baby Hamster Kidney cell. Alternatively, the cell is a prokayotic cell, e.g. a yeast cell or a bacterial cell.
The phrase xe2x80x9cin combination withxe2x80x9d refers to the administration of BPI protein with protein C either simultaneously, sequentially or a combination thereof. The BPI protein utilized and the appropriate dose level is known in the art and described in U.S. Pat. No. 5,756,464, herein incorporated by reference. A skilled artisan recognizes the appropriate dose level to use to achieve a pharmaceutically effective amount for treating sepsis. Pharmaceutically effective compositions comprising BPI protein may be administered systemically or topically. Systemic routes of administration include, intravenous, intramuscular or subcutaneous injection (including into a depot for long-term release), intraocular and retrobulbar, intrathecal, intraperitoneal (e.g. by intraperitoneal lavage), intrapulmonary using aerosalised-or nebulized drug, or transdermal. The preferred route is intravenous administration. When given parenterally, BPI protein compositions are generally injected in doses ranging from about 0.04 ug/kg/hr to about 4 mg/kg/hr. Preferably, the BPI protein is administered at about 4 ug/kg/hr to about 420 ug/kg/hr. More preferably the BPI protein is administered at about 50 ug/kg/hr to about 300 ug/kg/hr. Even more preferably the BPI protein is administered at about 100 ug/kg/hr to about 200 ug/kg/hr. The treatment may continue by continuous infusion or intermittent injection or infusion, at the same, reduced or increased dose per day for, e.g. 24 hours to 240 hours, and additionally as determined by the treating physician. BPI protein is preferably administered intravenously by an initial bolus injection followed by a continuous infusion. A preferred dosing regimen is about 0.1 mg/kg to about 10 mg/kg intravenous bolus of BPI protein followed by intravenous infusion at about 4 ug/kg/hr to about 420 ug/kg/hr, continuing for up to 10 days. Those skilled in the art can readily optimize pharmaceutically effective dosages and administration regimens for therapeutic compositions comprising BPI protein, as determined by good medical practice and the clinical condition of the individual patient.
The combination of the endotoxin neutralization and the Gram-negative bactericidal activity of BPI protein with the anti-coagulation and anti-inflammation activities of aPC results in enhanced efficacy in treating sepsis. The synergy results in the ability to reduce the dosages of the agents in combination therapy.